Introduction/Background*Detection of pathogenic somatic variants in the exonuclease domain (EDM) of the POLE-gene is of prognostic importance because of the excellent clinical outcomes of POLE-mutated endometrial cancers (EC). It is hypothesised that patients with POLE-mutated EC benefit of treatment de-escalation. Since 8–10% of EC carry a pathogenic POLE-mutation, it is of strong clinical importance to accurately determine the presence of these mutations. In current practice POLE-status can only be determined by DNA-sequencing methods, e.g. Sanger or Next-Generation-Sequencing (NGS). These techniques require a molecular biologist for correct interpretation, are relatively time consuming, not widely available and/or expensive. Due to the long turnaround time, it can be challenging to fit POLE-testing in tight timelines of clinical practice. To overcome this we have developed, and are in the process of validating, a rapid, simple, reliable and low-cost quantitative polymerase-chain-reaction (qPCR) assay for pathogenic POLE-mutations.
Methodology Primer and fluorescence-labelled 5’-nuclease probe-sequences of the five most frequently occurring pathogenic variations within exons 9, 13 and 14 of the POLE EDM have been developed and optimized using DNA extracted from formalin-fixed paraffin-embedded tumour tissues. The simplicity of the design enables POLE-status assessment within 4 hours. Ongoing calibration studies are evaluating the minimal amount and quality of DNA required.
Result(s)*Cut offs for failed, POLE-negative and -positive results were predefined based on 50 POLE-wildtypes and 6 POLE-mutated cases. In the range of uncertainty in between, NGS-testing is recommended. In our first testing set of 115 cases (40 POLE-mutated, 75 POLE-wildtype), two samples failed, (one POLE-mutant, one wildtype) possibly due to low DNA quality. Four samples fell in the range of uncertainty (three POLE-mutant, one wildtype). Almost all POLE-mutants (33/36) were positive with no false-positives. Sensitivity and specificity were 92% (95%CI 83%–100%) and 100%. Post-hoc adjusment of the lower cut-off yielded a sensitivity of 97% (95%CI 92%–100%) and specifity of 100%.
Conclusion*With this qPCR assay we developed a faster and simple alternative for targeted NGS-sequencing of the five most common pathogenic POLE-mutations. The assay–s simplicity in design and methods will make universal low-cost POLE-testing available for all EC patients.
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